Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling.

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

An expert discussion on the atypical hemolytic uremic syndrome nomenclature-identifying a road map to precision: a report of a National Kidney Foundation Working Group.

The term atypical hemolytic uremic syndrome has been in use since the mid-1970s. It was initially used to describe the familial or sporadic form of hemolytic uremic syndrome as opposed to the epidemic, typical form of the disease. Over time, the atypical hemolytic uremic syndrome term has evolved into being used to refer to anything that is not Shiga toxin-associated hemolytic uremic syndrome. The term describes a heterogeneous group of diseases of disparate causes, a circumstance that makes defining disease-specific natural history and/or targeted treatment approaches challenging. A working group of specialty-specific experts in the thrombotic microangiopathies was convened to review the validity of this broad term in an era of swiftly advancing science and targeted therapeutics. A Delphi approach was used to define and interrogate some of the key issues related to the atypical hemolytic uremic syndrome nomenclature.

Development of a target concentration intervention to individualize paroxysmal nocturnal hemoglobinuria treatment with pegcetacoplan.

Pegcetacoplan (Aspaveli®/Empaveli™) is a factor C3 inhibitor that is approved for the treatment of paroxysmal nocturnal hemoglobinuria. An individualized dosing strategy might be useful to improve patient-friendliness and cost-effectiveness of this very expensive drug. Therefore, the aim of this study was to develop an individualized treatment regimen for pegcetacoplan based on the pharmacokinetic-pharmacodynamic data of the manufacturer. We conducted a clinical trial simulation with the approved dosing regimen of 1080 mg twice-weekly and a target concentration intervention-based dosing regimen in patients with and without prior eculizumab use. For eculizumab-naïve patients, the target concentration intervention-based dosing regimen resulted in a comparable fraction of patients with LDH normalization (LDH < 226 U/L) and hemoglobulin normalization (> 12 g/dL) compared to the approved regimen (LDH 50.2% and 50.0% respectively and hemoglobulin 45.6% and 44.4%). A modest dose reduction of ~ 5% was possible with target concentration intervention-based dosing. An intensified dosing interval was necessary in 2.3% of the patients however an interval prolongation was possible in 28.2% of the patients. Similar results were obtained for patients prior treated with eculizumab. In this study we show the potential of an individualized dosing regimen of pegcetacoplan with can improve patient friendliness in approximately 30% of the patients and improve therapy in approximately 2% of the patients at slightly reduced costs.

Mutations in Genes Encoding Subunits of the RNA Exosome as a Potential Novel Cause of Thrombotic Microangiopathy.

Thrombotic microangiopathy (TMA) in association with RNA exosome encoding mutations has only recently been recognized. Here, we present an infant (female) with an EXOSC5 mutation (c.230_232del p.Glu77del) associated with the clinical phenotype known as CABAC syndrome (cerebellar ataxia, brain abnormalities, and cardiac conduction defects), including pontocerebellar hypoplasia, who developed renal TMA. At the age of four months, she presented with signs of septic illness, after which she developed TMA. A stool culture showed rotavirus as a potential trigger. The patient received eculizumab once, alongside supportive treatment, while awaiting diagnostic analysis of TMA, including genetic complement analysis, all of which were negative. Eculizumab was withdrawn and the patient's TMA recovered quickly. A review of the literature identified an additional four patients (age < 1 year) who developed TMA after a viral trigger in the presence of mutations in EXOSC3. The recurrence of TMA in one of these patients with an EXOSC3 mutation while on eculizumab treatment underscores the apparent lack of responsiveness to C5 inhibition. In conclusion, mutations in genes influencing the RNA exosome, like EXOSC3 and EXOSC5, characterized by neurodevelopment and neurodegenerative disorders could potentially lead to TMA in the absence of complement dysregulation. Hence, these patients were likely non-responsive to eculizumab.

Proposal for individualized dosing of eculizumab in atypical haemolytic uraemic syndrome: patient friendly and cost-effective.

BACKGROUND: Eculizumab is a lifesaving yet expensive drug for atypical haemolytic uraemic syndrome (aHUS). Current guidelines advise a fixed-dosing schedule, which can be suboptimal and inflexible in the individual patient. METHODS: We evaluated the pharmacokinetics (PK) and pharmacodynamics (PD) [classical pathway (CP) activity levels] of eculizumab in 48 patients, consisting of 849 time-concentration data and 569 CP activity levels. PK-PD modelling was performed with non-linear mixed-effects modelling. The final model was used to develop improved dosing strategies. RESULTS: A PK model with parallel linear and non-linear elimination rates best described the data with the parameter estimates clearance 0.163 L/day, volume of distribution 6.42 L, maximal rate 29.6 mg/day and concentration for 50% of maximum rate 37.9 mg/L. The PK-PD relation between eculizumab concentration and CP activity was described using an inhibitory Emax model with the parameter estimates baseline 101%, maximal inhibitory effect 95.9%, concentration for 50% inhibition 22.0 mg/L and  Hill coefficient 5.42. A weight-based loading dose, followed by PK-guided dosing was found to improve treatment. On day 7, we predict 99.95% of the patients to reach the efficacy target (CP activity <10%), compared with 94.75% with standard dosing. Comparable efficacy was predicted during the maintenance phase, while the dosing interval could be prolonged in ∼33% of the population by means of individualized dosing. With a fixed-dose 4-week dosing interval to allow for holidays, treatment costs will increase by 7.1% and we predict 91% of the patients will reach the efficacy target. CONCLUSIONS: A patient-friendly individualized dosing strategy of eculizumab has the potential to improve treatment response at reduced costs.

Dose optimalization of subcutaneous ravulizumab is predicted to yield significant savings and to improve patient friendliness.

Ravulizumab is an expensive complement C5-inhibitor for the treatment of paroxysmal nocturnal haemoglobinuria. Recently, a subcutaneous formulation has entered the market, for which the approved dosing regimen results in supratherapeutic ravulizumab concentrations in the majority of patients in the registration studies. Therefore, we explored alternative dosing regimens in silico based on the registration data of the manufacturer. Extending the interval from 1 to 2 weeks or individualized dosing based on therapeutic drug monitoring resulted in therapeutic ravulizumab concentrations and comparable predicted efficacy in terms of lactate dehydrogenase normalization, with dose reductions up to 64%. We here show that with an individualized dose, a substantial dose reduction for subcutaneous ravulizumab might be possible with improved patient-friendliness.

The Role of Properdin in C5 Convertase Activity and C5b-9 Formation in the Complement Alternative Pathway.

The complement system is an important part of innate immunity. Complement activation leads to formation of convertase enzymes, switch of their specificity from C3 to C5 cleavage, and generation of lytic membrane attack complexes (C5b-9) on surfaces of pathogens. Most C5 cleavage occurs via the complement alternative pathway (AP). The regulator properdin promotes generation and stabilization of AP convertases. However, its role in C5 activation is not yet understood. In this work, we showed that serum properdin is essential for LPS- and zymosan-induced C5b-9 generation and C5b-9-mediated lysis of rabbit erythrocytes. Furthermore, we demonstrated its essential role in C5 cleavage by AP convertases. To this end, we developed a hemolytic assay in which AP convertases were generated on rabbit erythrocytes by using properdin-depleted serum in the presence of C5 inhibitor (step 1), followed by washing and addition of purified C5-C9 components to allow C5b-9 formation (step 2). In this assay, addition of purified properdin to properdin-depleted serum during convertase formation (step 1) was required to restore C5 cleavage and C5b-9-mediated hemolysis. Importantly, C5 convertase activity was also fully restored when properdin was added together with C5b-9 components (step 2), thus after convertase formation. Moreover, with C3-depleted serum, not capable of forming new convertases but containing properdin, in step 2 of the assay, again full C5b-9 formation was observed and blocked by addition of properdin inhibitor Salp20. Thus, properdin is essential for the convertase specificity switch toward C5, and this function is independent of properdin's role in new convertase formation.

Protective mechanisms harnessing against injurious heme and preventing kidney damage in STEC-HUS: toward new therapies?

Hemolytic uremic syndrome can be initiated by Escherichia coli infections (Shiga-toxin-producing enterohemorrhagic Escherichia coli hemolytic uremic syndrome). When hemoglobin and heme released from ruptured erythrocytes interact with the kidney cells, this can result in platelet activation, vascular inflammation and occlusion, and kidney injury. Pirschel et al. now report that in the absence of protective mechanisms against free hemoglobin and heme, heme-induced kidney injury can be exacerbated. Therapeutic strategies should therefore also target heme-mediated deleterious effects in (severely ill) patients with Shiga-toxin-producing enterohemorrhagic Escherichia coli hemolytic uremic syndrome.

Long-term follow-up including extensive complement analysis of a pediatric C3 glomerulopathy cohort.

BACKGROUND: C3 glomerulopathy (C3G) is a rare kidney disorder characterized by predominant glomerular depositions of complement C3. C3G can be subdivided into dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). This study describes the long-term follow-up with extensive complement analysis of 29 Dutch children with C3G. METHODS: Twenty-nine C3G patients (19 DDD, 10 C3GN) diagnosed between 1992 and 2014 were included. Clinical and laboratory findings were collected at presentation and during follow-up. Specialized assays were used to detect rare variants in complement genes and measure complement-directed autoantibodies and biomarkers in blood. RESULTS: DDD patients presented with lower estimated glomerular filtration rate (eGFR). C3 nephritic factors (C3NeFs) were detected in 20 patients and remained detectable over time despite immunosuppressive treatment. At presentation, low serum C3 levels were detected in 84% of all patients. During follow-up, in about 50% of patients, all of them C3NeF-positive, C3 levels remained low. Linear mixed model analysis showed that C3GN patients had higher soluble C5b-9 (sC5b-9) and lower properdin levels compared to DDD patients. With a median follow-up of 52 months, an overall benign outcome was observed with only six patients with eGFR below 90 ml/min/1.73 m2 at last follow-up. CONCLUSIONS: We extensively described clinical and laboratory findings including complement features of an exclusively pediatric C3G cohort. Outcome was relatively benign, persistent low C3 correlated with C3NeF presence, and C3GN was associated with higher sC5b-9 and lower properdin levels. Prospective studies are needed to further elucidate the pathogenic mechanisms underlying C3G and guide personalized medicine with complement therapeutics.

The potential of individualized dosing of ravulizumab to improve patient-friendliness of paroxysmal nocturnal haemoglobinuria treatment at reduced costs.

Ravulizumab is a very expensive complement C5-inhibitor for the treatment of paroxysmal nocturnal haemoglobinuria, with a fixed-dosing interval of 8 weeks. For lifelong treatment, a cost-effective and patient-friendly dosing strategy is preferred. We therefore explored alternative ravulizumab dosing regimens in silico based on the thorough dose-finding studies of the manufacturer. Extending the interval to 10 weeks or individually extending the interval to a mean of 12.8 weeks based on pharmacokinetic monitoring resulted in noninferior efficacy in terms of lactate dehydrogenase normalization, with drug cost savings up to 37%. We here show the potential of individualized ravulizumab dosing to improve patient-friendliness at reduced costs.

Cell Biological Responses after Shiga Toxin-1 Exposure to Primary Human Glomerular Microvascular Endothelial Cells from Pediatric and Adult Origin.

Hemolytic uremic syndrome (HUS) is characterized by a triad of symptoms consisting of hemolytic anemia, thrombocytopenia and acute renal failure. The most common form of HUS is caused by an infection with Shiga toxin (Stx) producing Escherichia coli bacteria (STEC-HUS), and the kidneys are the major organs affected. The development of HUS after an infection with Stx occurs most frequently in children under the age of 5 years. However, the cause for the higher incidence of STEC-HUS in children compared to adults is still not well understood. Human glomerular microvascular endothelial cells (HGMVECs) isolated and cultured from pediatric and adult kidney tissue were investigated with respect to Stx binding and different cellular responses. Shiga toxin-1 (Stx-1) inhibited protein synthesis in both pediatric and adult HGMVECs in a dose-dependent manner at basal conditions. The preincubation of pediatric and adult HGMVECs for 24 hrs with TNFα resulted in increased Stx binding to the cell surface and a 20-40% increase in protein synthesis inhibition in both age groups. A decreased proliferation of cells was found when a bromodeoxyuridine (BrdU) assay was performed. A trend towards a delay in endothelial wound closure was visible when pediatric and adult HGMVECs were incubated with Stx-1. Although minor differences between pediatric HGMVECs and adult HGMVECs were found in the assays applied in this study, no significant differences were observed. In conclusion, we have demonstrated that in vitro primary HGMVECs isolated from pediatric and adult kidneys do not significantly differ in their cell biological responses to Stx-1.

Preparing for a kidney transplant: Medical nephrectomy in children with nephrotic syndrome.

Nephrotic syndrome is characterized by proteinuria, hypoalbuminemia, and general edema. These symptoms may persist in children who reach ESRD, which is unfavorable for the patient's allograft outcome. In addition, this may hamper early diagnosis of a relapse after transplantation. Surgical bilateral nephrectomy is often considered for that reason, but medical nephrectomy may be a less invasive alternative. In this retrospective single-center case series, we identified all children on dialysis with ESRD due to nephrotic syndrome in which a medical nephrectomy was attempted before kidney transplantation between 2013 and 2018. Outcome was measured by urine output and serum albumin levels. Eight patients with either congenital nephrotic syndrome or focal segmental glomerular sclerosis were included in the study. All patients received an ACE inhibitor as drug of first choice for medical nephrectomy, to which 5 patients responded with oligoanuria and a significant rise in serum albumin, and 3 patients responded insufficiently. In 1 of these 3 patients, diclofenac was added to the ACE inhibitor, with good result. In the other 2 patients, indomethacin was initiated without success, and surgical bilateral nephrectomy was performed. Overall, 6/8 patients had a successful medical nephrectomy and did not need surgical nephrectomy. No recurrence of nephrotic syndrome was found after kidney transplantation in all but one. Medical nephrectomy with ACE inhibitors and/or non-steroidal anti-inflammatory drugs is a safe and non-invasive therapy to minimize proteinuria in children with ESRD due to nephrotic syndrome before kidney transplantation. We suggest that this strategy should be considered as therapy before proceeding with surgical nephrectomy.

Treatment-resistant nephrotic syndrome in dense deposit disease: complement-mediated glomerular capillary wall injury?

BACKGROUND: The C3 glomerulopathies (C3G) are recently defined glomerular diseases, attributed to abnormal complement regulation. Dense deposit disease (DDD) is part of the spectrum of C3G, characterized by electron-dense deposits in the lamina densa of the glomerular basement membrane. Patients with DDD present with hematuria, variable degrees of proteinuria, and kidney dysfunction. Kidney biopsies typically disclose proliferative and inflammatory patterns of injury. Treatment with glucocorticoids and mycophenolate mofetil has been shown to achieve remission of proteinuria in a significant proportion of C3G patients. CASE-DIAGNOSIS/TREATMENT: We report two patients with persistent nephrotic syndrome while on immunosuppressive therapy. Repeat kidney biopsies disclosed massive C3 deposits with foot process effacement in the absence of proliferative or inflammatory lesions on light microscopy. CONCLUSION: These cases, coupled with data from animal models of disease and the variable response to eculizumab in C3G patients, illustrate that two different pathways might be involved in the development of kidney injury in C3G: a C5-independent pathway leading to glomerular capillary wall injury and the development of proteinuria versus a C5-dependent pathway that causes proliferative glomerulonephritis and kidney dysfunction.

Correction to: Eculizumab in atypical hemolytic uremic syndrome: strategies toward restrictive use.

The original version of this article unfortunately contained two mistakes. The presentation of Table 1 and Fig. 1 was incorrect. The corrected versions are given below.

Eculizumab in atypical hemolytic uremic syndrome: strategies toward restrictive use.

With the introduction of the complement C5-inhibitor eculizumab, a new era was entered for patients with atypical hemolytic uremic syndrome (aHUS). Eculizumab therapy very effectively reversed thrombotic microangiopathy and reduced mortality and morbidity. Initial guidelines suggested lifelong treatment and recommended prophylactic use of eculizumab in aHUS patients receiving a kidney transplant. However, there is little evidence to support lifelong therapy or prophylactic treatment in kidney transplant recipients. Worldwide, there is an ongoing debate regarding the optimal dose and duration of treatment, particularly in view of the high costs and potential side effects of eculizumab. An increasing but still limited number of case reports and small cohort studies suggest that a restrictive treatment regimen is feasible. We review the current literature and focus on the safety and efficacy of restrictive use of eculizumab. Our current treatment protocol is based on restrictive use of eculizumab. Prospective monitoring will provide more definite proof of the feasibility of such restrictive treatment.

The role of properdin in complement-mediated renal diseases: a new player in complement-inhibiting therapy?

Properdin is known as the only positive regulator of the complement system. Properdin promotes the activity of this defense system by stabilizing its key enzymatic complexes: the complement alternative pathway (AP) convertases. Besides, some studies have indicated a role for properdin as an initiator of complement activity. Though the AP is a powerful activation route of the complement system, it is also involved in a wide variety of autoimmune and inflammatory diseases, many of which affect the kidneys. The role of properdin in regulating complement in health and disease has not received as much appraisal as the many negative AP regulators, such as factor H. Historically, properdin deficiency has been strongly associated with an increased risk for meningococcal disease. Yet only recently had studies begun to link properdin to other complement-related diseases, including renal diseases. In the light of the upcoming complement-inhibiting therapies, it is interesting whether properdin can be a therapeutic target to attenuate AP-mediated injury. A full understanding of the basic concepts of properdin biology is therefore needed. Here, we first provide an overview of the function of properdin in health and disease. Then, we explore its potential as a therapeutic target for the AP-associated renal diseases C3 glomerulopathy, atypical hemolytic uremic syndrome, and proteinuria-induced tubulointerstitial injury. Considering current knowledge, properdin-inhibiting therapy seems promising in certain cases. However, knowing the complexity of properdin's role in renal pathologies in vivo, further research is required to clarify the exact potential of properdin-targeted therapy in complement-mediated renal diseases.

Glyco-iELISA: a highly sensitive and unambiguous serological method to diagnose STEC-HUS caused by serotype O157.

BACKGROUND: Providing proof of presence of Shiga toxin-producing E. coli (STEC) infection forms the basis for differentiating STEC-hemolytic uremic syndrome (HUS) and atypical HUS. As the gold standard to diagnose STEC-HUS has limitations, using ELISA to detect serum antibodies against STEC lipopolysaccharides (LPS) has proven additional value. Yet, conventional LPS-ELISA has drawbacks, most importantly presence of cross-reactivity due to the conserved lipid A part of LPS. The newly described glyco-iELISA tackles this issue by using modified LPS that eliminates the lipid A part. Here, the incremental value of glyco-iELISA compared to LPS-ELISA is assessed. METHODS: A retrospective study was performed including all pediatric patients (n = 51) presenting with a clinical pattern of STEC-HUS between 1990 and 2014 in our hospital. Subsequently, the diagnostic value of glyco-iELISA was evaluated in a retrospective nationwide study (n = 264) of patients with thrombotic microangiopathy (TMA). LPS- and glyco-iELISA were performed to detect IgM against STEC serotype O157. Both serological tests were compared with each other and with fecal diagnostics. RESULTS: Glyco-iELISA is highly sensitive and has no cross-reactivity. In the single-center cohort, fecal diagnostics, LPS-ELISA, and glyco-iELISA identified STEC O157 infection in 43%, 65%, and 78% of patients, respectively. Combining glyco-iELISA with fecal diagnostics, STEC infection due to O157 was detected in 89% of patients. In the nationwide cohort, 19 additional patients (8%) were diagnosed with STEC-HUS by glyco-iELISA. CONCLUSION: This study shows that using glyco-iELISA to detect IgM against STEC serotype O157 has clear benefit compared to conventional LPS-ELISA, contributing to optimal diagnostics in STEC-HUS.